Cells are continually exposed to factors, such as intracellular reactive species and environmental agents, which are capable of causing DNA damage. The potentially mutagenic consequences of DNA damage are minimized by DNA repair pathways, which are broadly characterized into three forms: base excision repair (BER), mismatch repair (MMR), and nucleotide excision repair (NER) (Wood et al., Science, 291: 1284-1289 (2001)). Deficiencies in DNA damage repair underlie the pathogenesis of cancer as well as many genetic disorders, such as Xeroderma pigmentosum, Cockayne syndrome, and Ataxia-telangiectasia.
Exposure to ultraviolet light (UV) irradiation or chemical mutagens leads to the accumulation of damaged DNA, which in turn, results in mutations that contribute to the development of cancer. Eukaryotic cells respond to UV irradiation by induction of the NER pathway, which identifies and removes damaged DNA, and by activation of the DNA damage checkpoint to halt cell cycle progression, thereby allowing time for NER action. NER is the major DNA repair pathway by which cells remove helix-distorting DNA damage caused by UV irradiation and chemical mutagens (Friedberg et al., DNA Repair and Mutagenesis, 2nd Edition, ASM Press, Washington, D.C. (2006)).
NER is a multistep process that employs over 30 proteins to carry out the distinct steps of recognizing DNA damage, incising the 5′ and 3′ ends of the lesion to remove damaged DNA, filling in the gap with DNA polymerase, and attaching the newly synthesized DNA to the parental DNA via DNA ligase activity (see, e.g., Friedberg et al., DNA Repair and Mutagenesis, 2nd Edition, ASM Press, Washington, D.C. (2006); Sancar, Annu. Rev. Biochem., 65: 43-81 (1996)). NER consists of two pathways with distinct DNA strand specificities: the transcription-coupled repair pathway (TCR) which removes lesions from DNA strands transcribed by RNA polymerase II, and the global genomic repair pathway (GGR) which repairs damage on the non-transcribed strand of expressed genes as well as from inactive chromatin (reviewed in Hanawalt, Oncogene, 21: 8949-8956 (2002)). The NER process has been studied extensively, and the components essential to perform the excision and repair reactions have been defined by in vitro reconstitution using recombinant proteins and damaged DNA templates (Aboussekhra et al., Cell, 80: 859-868 (1995); Araujo et al., Genes Dev., 14: 349-359 (2000); Mu et al., J. Biol. Chem., 271: 8285-8294 (1996); Mu et al., J. Biol. Chem., 270: 2415-2418 (1995)).
NER factors involved in the GGR pathway of DNA damage recognition include XPA-RPA, XPC-HR23B, and the heterodimeric, damage-specific DNA binding proteins consisting of DDB1 (p127) and DDB2 (p48) subunits. Among these DNA damage sensors, the heterodimeric DDB1-DDB2 exhibit the highest affinity (designated UV-DDB activity) for UV-induced cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) (Batty et al., J. Mol. Biol., 300: 275-290 (2000)). Mutations in DDB2 are responsible for xeroderma pigmentosum complementation group E (XP-E) cases, which are characterized by defects in GGR-mediated removal of damaged DNA and predisposition to skin cancer (Wittschieben and Wood, DNA Repair (Amst), 2: 1065-1069 (2003). Following UV irradiation, DDB1 and DDB2 immediately accumulate on damaged DNA and are subsequently ubiquitinated and degraded by the CUL4A ubiquitin ligase (Chen et al., Mol. Cell, 22: 489-499 (2006); Fitch et al., DNA Repair (Amst), 2: 819-826 (2003); Groisman et al., Cell, 113: 357-367 (2003); Rapic-Otrin et al., Nucleic Acids Res., 30: 2588-2598 (2002)). CUL4A is also responsible for the turnover of DDB2 under normal growth conditions (Chen et al., J. Biol. Chem., 276: 48175-48182 (2001); Nag et al., Mol. Cell. Biol., 21: 6738-6747 (2001), which leads to an overall decrease in UV-DDB activity (Chen et al., J. Biol. Chem., 276: 48175-48182 (2001)).
The CUL4A ubiquitin ligase functions as a component of a multimeric complex wherein the C-terminus of CUL4A interacts with the RING finger protein Rbx1/ROC1/Hrt1 (hereinafter referred to as Rbx1) to recruit the E2 ubiquitin-conjugating enzyme, and the N-terminus of CUL4A interacts with DDB1. DDB1, in turn, acts as an adaptor, binding to DDB1, CUL4A associated factors (DCAFs), which serve as specific substrate receptors (Angers et al., Nature, 443: 590-593 (2006); He et al., Genes Dev., 20: 2949-2954 (2006); Higa et al., Nat Cell Biol., 8: 1277-1283 (2006); Jin et al., Mol. Cell, 23: 709-721 (2006); Lee and Zhou, Mol. Cell, 26: 775-780 (2007)). CUL4B, the other CUL4 family member, has extensive sequence homology with CUL4A and shares some redundant functions with CUL4A, including maintaining cell growth and mediating the ubiquitination of certain CUL4 targets (Higa et al., Nat. Cell Biol., 5: 1008-1015 (2003); Hu et al., Nat. Cell Biol., 6: 1003-1009 (2004)). CUL4B containing ubiquitin ligase complexes have some unique features, such as the ability to degrade sex steroid hormone receptors (Ohtake et al., Nature, 446: 562-566 (2007)). Additionally, CUL4B mutations have been identified as the causal genetic defects underlying X-linked mental retardation (Tarpey et al., Am. J. Hum. Genet., 80: 345-352 (2007); Zou et al., Am. J. Hum. Genet., 80: 561-566 (2007)).
To date, most of the cancer therapies that target DNA repair pathways are substances that inhibit DNA repair in cancer cells in order to enhance the effects DNA-damaging chemotherapies and radiotherapies (Kelley and Fishel. Anticancer Agents Med. Chem., 8(4): 417-25 (2008)).
Comparatively fewer attempts have been made to improve or accelerate DNA repair in order to reduce the consequences of DNA damage after it has occurred in order to prevent or treat disease, although compositions comprising T4 endonuclease V have been examined as a potential therapy for skin cancer (Cafardi and Elmets. Expert Opin. Biol. Ther., 8(6): 829-38 (2008)).
Thus, there is a need for compositions and methods to enhance DNA repair in cells and in animals. This invention provides such compositions and methods, which may be useful for the prevention or treatment of diseases associated with DNA damage.